This application is a 371 national stage application of PCT/US96/04206, filed Mar. 27, 1996, which claims the benefit of priority to Israel Application No. 113,159, filed Mar. 28, 1995.
The present invention relates to synthetic peptides and to pharmaceutical compositions comprising them useful for the treatment of systemic lupus erythematosus (SLE) in humans.
Autoimmune diseases are characterized by immune responses that are directed against self antigens. These responses are maintained by the persistent activation of self-reactive T lymphocytes. T lymphocytes are specifically activated upon recognition of foreign and/or self antigens as a complex with self major histocompatibility complex (MHC) gene pro(ducts on the surface of antigen-presenting cells (APC).
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown origin and cure. Despite the extensive research on the mechanisms underlying the induction and development of SLE, the information available on the etiology of the disease is very limited due to the heterogeneity of SLE patients on one hand, and the lack of an experimental model in which the induction of the disease could be controlled, on the other hand.
The cause of SLE is unknown and it has heterogeneous clinical manifestations. Furthermore, no specific treatment aimed towards the prevention or cure of SLE is available. Despite the extensive research on the mechanisms underlying the induction of SLE, the information on the etiology of the disease is very limited. Studies on SLE have been performed until recently using peripheral blood lymphocytes (PBL) of patients at different clinical stages and under various treatment protocols. Alternatively, murine strains that develop spontaneous SLE-like disease were investigated as a model for SLE. This kind of analysis led to incomplete and confusing interpretations of the role of various immunological and non-immunological factors in either inducing or sustaining the disease, mainly due to the heterogeneity of patients on one hand and the inability to control the induction phase of the disease in murine SLE strains on the other hand.
Several years ago, an animal model of SLE has been established in the laboratory of one of the present inventors. This model, based on the concept of the idiotypic network, developed a wide spectrum of lupus-related autoantibodies and clinical manifestations (Mendlovic et al., 1988). The induction was carried out by the immunization of mouse strains that do not develop any spontaneous autoimmune disorders, with a human anti-DNA monoclonal antibody (mAb) which bears a common idiotype termed 16/6 Id (Shoenfeld et al., 1983). Following immunization, the mice produced antibodies specific to the 16/6 Id, antibodies that bear the 16/6 Id and antibodies directed against different nuclear antigens (dsDNA, ssDNA, Sm, ribonucleoprotein (RNP), Ro, La and others). The serological findings were associated with leukopenia, elevated erythrocyte sedimentation rate, proteinuria, abundance of immune complexes in the kidneys and sclerosis of the glomeruli (Mendlovic et al., 1988), which are typical manifestations of SLE. The present inventors have further shown that the experimental disease could be induced by a murine anti-16/6 Id mAb (Mendlovic et al., 1989) and by the mouse anti-anti 16/6 Id (16/6 Id+) mAb (Waisman et al., 1993). The induction of the disease is genetically controlled, and thus is strain dependent (Mendlovic et al., 1990). This unique model for the induction of experimental SLE provides the appropriate tools to clearly dissect the different steps and the linked immune parameters involved in the induction and development of SLE.
SLE is a systemic autoimmune disease characterized by the formation of autoantibodies against self-antigens, such as DNA, Sm, Ro, La, RNP, cardiolipin and histones. The etiology of SLE is unknown, and understanding the mechanism by which these self-antibodies arise might provide insight to this problem. For this purpose, the present inventors have produced a variety of monoclonal autoantibodies derived from C3H.SW mice in which experimental SLE was induced. As a rule, the monoclonal autoantibodies that were capable of eliciting antibodies that bear the 16/6 Id or react with it were found to be pathogenic and thus capable of inducing experimental SLE (Fricke et al., 1990; Sthoeger et al., 1993). Later on, the variable (V) regions of nine autoantibodies-that bind either DNA or HeLa nuclear extract (NE), isolated from the C3H.SW mice with experimental SLE, were sequenced (Waisman and Mozes, 1993). Monoclonal antibodies with different specificity were analyzed in an attempt to determine the connections between the different autoantibodies. Three mAb were found to bind DNA, and were shown to exhibit sequence characteristics of pathogenic anti-DNA antibodies. One of these mAb, designated 2C4C2, was shown to use a heavy (H) chain V region gene (VH) identical to the VH of anti-DNA mAb isolated from other lupus-prone mice, namely (NZBxc3x97NZW)F1. The light (L) chain V region gene (VL) of mAb 2C4C2 is 98% homologous to the VL of another anti-DNA mAb, also isolated from (NZBxc3x97NZW)F1 mice. The other two anti-DNA mAb, designated 5G12-4 and 5G12-6, share 93% of their VH sequences with that of mAb 2C4C2. Six mAb bound proteins of HeLa NE. The nine mAb use a total of five VH and four VL germ-line genes, demonstrating that the autoantibodies induced in mice with experimental SLE do not originate from one B cell clone. Three of the nine VH and VL were identical in sequence to germ-line genes, while at least three others had somatic mutations. The latter suggests that these autoantibodies arise in mice by both usage of existing (pre-immune) B cells, and through an antigen-driven process. Furthermore, it appears that autoantibodies found in mice with experimental SLE use genetic elements similar to those used by mAb that were isolated from mouse strains which develop lupus spontaneously.
T cells play an important role in the induction and development of experimental SLE. Thus, T cell lines and clones specific to the 16/6 Id were shown to induce experimental SLE in syngeneic recipients similarly to the 16/6 antibody. Therefore, following the inoculation of the activated cells of the lines, the mice developed both the serology and the renal damage which is typical to SLE (Fricke et al., 1991). Furthermore, a 16/6 Id specific T cell line of C3H.SW origin induced SLE in C57BL/6 mice that were shown to be resistant to the induction of the disease following injections with either the 16/6 Id or the anti-16/6 Id mAb (Mendlovic et al., 1990).
In an attempt to identify the pathogenic region of the 16/6 Id, (Fabxe2x80x2)2 fragments were prepared of the 16/6 Id mAb and were found to retain the specificity and pathogenic capacity of the whole 16/6 Id molecule (Ruiz et al., 1994).
The mAb 5G12 that was isolated from mice with experimental SLE and was shown to bind DNA and bear the 16/6 Id, is capable of inducing experimental SLE in mice (Waisman et al., 1993). T cells that react specifically to mAb by proliferation, are probably reacting to peptides representing sequences from their complementarity-determining regions (CDR). It is very likely that the T cells recognize the V regions of the above antibodies since they do not react with other antibodies that carry the same constant region but have different specificities. Within the variable region, the regions with the highest probability to be recognized are the CDR, since those are the regions that differ the most between the various antibodies. The CDR regions of the VH sequences of the nine pathogenic murine mAb mentioned above that induce SLE in mice, are boxed in FIG. 1 of Waisman and Mozes, 1993, in which the complete nucleotide and deduced amino acid sequences for the VH of the nine mAb are presented.
It is an object of the present invention to provide means for specific treatment of patients with SLE.
For this purpose, the invention provides peptides and analogs thereof based on the CDR regions of pathogenic monoclonal autoantibodies isolated from mice with experimental SLE.
Thus, in one aspect, the invention relates to a synthetic peptide selected from the group consisting of:
(i) a peptide of at least 12 and at most 30 amino acid residues based on a complementarity-determining region (CDR) of the heavy or light chain of a pathogenic anti-DNA monoclonal antibody that induces a systemic lupus erythematosus (SLE)-like disease in mice (hereinafter CDR-based peptide), a salt or a chemical derivative thereof,
(ii) an analog of a CDR-based peptide defined in (i), a salt or a chemical derivative thereof;
(iii) a dual synthetic peptide comprising two such peptides of (i) or analogs of (ii) covalently linked to one another either directly or through a short linking chain;
(iv) a peptide polymer comprising a plurality of sequences of said peptide (i) or analog thereof (ii); and
(v) a peptide polymer (iv) attached to a macromolecular carrier.
In one embodiment of this aspect, the synthetic peptide is capable of:
(i) inhibiting specifically the proliferative response and cytokine secretion of T lymphocytes of mice that are high responders to SLE-inducing autoantibodies; or
(ii) inhibiting development of SLE in mice that are susceptible to SLE-induction by pathogenic autoantibodies.
The synthetic peptides and analogs thereof according to the invention may be selected from the group consisting of peptides having the sequences I to V herein, wherein:
(i) the peptide of sequence I has the formula:
T G Y Y X1 X2 X3 X4 X5 Q S P E K S L E W I G (SEQ. ID NO:1) [I]
wherein X1 is Met, Ala or Val; X2 is Gln, Asp, Glu or Arg; X3 is Trp or Ala; X4 is Val or Ser; and X5 is Lys, Glu or Ala;
(ii) the peptide of sequence II has the formula:
E I N P S T G G X6 X7 X8 X9X10 X11 X12 K A K A T (SEQ ID NO:2) [II]
wherein X6 and X7 are each Thr, Val or Ala; X8 is Tyr or Phe; X9 is Asn or Asp; X10 is Gln or Glu; X11 is Lys or Glu, and X12 is Phe or Tyr;
(iii) the peptide of sequence III has the formula:
Y Y C A R X13X14 X15 X16 P Y A X17 X18 Y W G Q G S (SEQ ID NO:3) [III]
wherein X13 is Phe, Thr or Gly; X14 is Leu, Ala or Ser; X15 is Trp or Ala; X16 is Glu or Lys; X17 is Met or Ala, and X18 is Asp, Lys or Ser;
(iv) the peptide of sequence IV has the formula:
G Y N X19 X20 X21 X22 X23 X24 S H G X25 X26 L E W I G (SEQ ID NO:4) [IV]
wherein X19 is Met or Ala; X20 is Asn, Asp or Arg; X21 is Trp or Ala; X22 is Val or Ser; X23 is Lys or Glu; X24 is Gln or Ala; X25 is Lys or Glu, and X26 is Ser or Ala; and
(v) the peptide of sequence V has the formula:
Y Y C A R X27 X28 X29 Y G X30 X31 X32 G Q G T L (SEQ ID NO:5) [V]
wherein X27 is Ser or Phe; X28 is Gly or Ala; X29 is Arg, Ala or Glu; X30 is Asn or Asp; X31 is Tyr or Phe, and X32 is Trp, His or Ala.
In preferred embodiments, peptides I to V have the sequences Ia-Va herein:
T G Y Y M Q W V K Q S P E K S L E W I G (SEQ ID NO:6) (Ia)
E I N P S T G G T T Y N Q K F K A K A T (SEQ ID NO:7) (IIa)
Y Y Y C A R F L W E P Y A M D Y W G Q G S (SEQ ID NO:8) (IIIa)
G Y N M N W V K Q S H G K S L E W I G (SEQ ID NO:9) (IVa)
Y Y C A R S G R Y G N Y W G Q G T L (SEQ ID NO:10) (Va)
Peptides Ia to IIIa are based on the CDR1, CDR2 and CDR3 regions, respectively, of the VH chain of mAb 5G12, and peptides IVa and Va are based on the CDR1 and CDR3 regions, respectively, of the VH chain of mAb 2C4C2 (Waisman and Mozes, 1993).
In another aspect, the invention relates to pharmaceutical compositions for the treatment of SLE comprising a synthetic peptide or peptide polymer of the invention and a pharmaceutically acceptable carrier.
In still another aspect, the invention relates to a method of treatment of a SLE patient comprising administering to a SLE patient an effective amount of a synthetic peptide or peptide polymer of the invention.